Inherited ARPC5 mutations cause an actinopathy impairing cell motility and disrupting cytokine signaling

We describe the first cases of germline biallelic null mutations in ARPC5, part of the Arp2/3 actin nucleator complex, in two unrelated patients presenting with recurrent and severe infections, early-onset autoimmunity, inflammation, and dysmorphisms. This defect compromises multiple cell lineages and functions, and when protein expression is reestablished in-vitro, the Arp2/3 complex conformation and functions are rescued. As part of the pathophysiological evaluation, we also show that interleukin (IL)−6 signaling is distinctively impacted in this syndrome. Disruption of IL-6 classical but not trans-signaling highlights their differential roles in the disease and offers perspectives for therapeutic molecular targets.


Case reports
Patient 1 (P1) was the eldest daughter born to healthy consanguineous parents of Lebanese descent . She was born at 41 weeks gestation with intrauterine growth restriction. P1 became symptomatic within the first months of life with gastrointestinal and respiratory tract bleeding, hepatosplenomegaly, Evans syndrome (autoimmune hemolytic anemia and thrombocytopenia) and neutrophilia. Her clinical course was remarkable for respiratory tract complications including recurrent/severe viral, bacterial, and fungal infections, pulmonary alveolar proteinosis and pneumatoceles; eczema; juvenile dermatomyositis-like focal myositis; delayed wound healing; recurrent episodes of paralytic ileus; celiac disease; hepatitis; Heterozygous, unaffected Homozygous, affected Genotype unknown, affected crosses, from (+) to (+++) indicate less to more severe phenotypes, respectively. c Patient 1 images. In the left image, a standing radiograph of P1 shows biconvex thoracolumbar scoliosis with convex right thoracic spinal curvature and convex left thoracolumbar spinal curvature; a right upper lobe pneumatocele is also identified. In the right images (upper, middle and lower, respectively), lung ground glass opacities, multiple abdominal scars product of abnormal wound healing, and right sided myositis of the thigh, are also detected. minimal change disease nephropathy; short stature, facial dysmorphisms and scoliosis. Multiple autoantibodies were detected at various timepoints of her life; acute phase reactants, particularly C-reactive protein (CRP), were persistently elevated. Immunosuppressive and immunomodulatory treatments (e.g., steroids, rituximab, and high dose intravenous immunoglobulins) were used with partial and unsustained success. At 15 years of age, she died after a sudden episode of hemoptysis and hematochezia; an autopsy was not performed. Biospecimens from P1 were available for research studies. Patient 2 (P2) was the youngest daughter born to healthy consanguineous Iranian parents (Fig. 1a, b). She became symptomatic since early in life when she presented with omphalitis. Her clinical course was remarkable for respiratory tract complications including severe/recurrent bacterial infections and pneumatoceles as well as skin infections, poor wound healing, multifocal aseptic bone lesions, and scoliosis. Neurodevelopmental delay, spasticity and brain atrophy were also evidenced. Acute phase reactants were persistently elevated. At the age of one year, she died because of progressive neurologic and respiratory disease; an autopsy was not performed. Cells from P2 were not available for research studies. P2's elder sister presented with severe, invasive bacterial infections (i.e., Staphylococcus aureus and Pseudomonas aeruginosa) since the neonatal period; hepatosplenomegaly and hematochezia were also present, but no neurologic manifestations were observed. The patient died at six months of age due to progressive perineal necrotic infectious complications; an autopsy was not performed. Biospecimens from this patient were not available for testing.

P1 and P2 detailed clinical descriptions are available as a Supplementary Note
Clinical laboratory evaluation on P1 and P2 revealed anemia and elevated white blood cell counts, including neutrophilia, and lympho-monocytosis (Fig. 1b Supplementary Fig. 1). B cells (prior to anti-CD20 treatment) were increased, with high frequency of age-associated B cells, a subset linked to autoimmunity 12 (Supplementary Table 2 and Supplementary Fig. 2). NK cells fell within normal ranges, but NKT cells were elevated. These mononuclear cell changes were complemented by transcriptional/functional defects as determined by single cell differentially expressed gene analysis (Fig. 2, Supplementary Table 2). T-cell proliferation was normal in response to phytohemagglutinin and T-cell receptor (TCR) stimulation ( Supplementary Fig. 1). Specific antibody responses to recall protein were normal but unsustained to polysaccharide antigens (Supplementary Table 1). B cells proliferated normally upon various stimulating conditions ( Supplementary Fig. 2). IgG, IgA, and IgM serum levels were normal/high in P1 and P2; IgE was elevated in P2 (Supplementary Table 1).

Candidate variant selection
Whole exome sequencing trio analysis was performed in two families with unknown genetic diseases and phenotypic similarities (Fig. 1a-  ; CADD score of 30; not found in gnomAD) was also detected in P2, and possibly associated with her neurologic manifestations.

Mutant ARPC5 biochemical and functional studies
While the mutant ARPC5 mRNA was detected ( Supplementary Fig. 5), expression of ARPC5 was not detected in peripheral blood mononuclear cells (PBMC), T-cell blasts, and fibroblasts from P1 (Fig. 3a, Supplementary Fig. 6). N-terminus truncated proteins, resulting from alternative transcription initiation sites were not detected either. The heterozygous parents showed intermediate expression levels of ARPC5. Immunoblotting of the other Arp2/3 complex subunits revealed reduced expression of ARPC1A and ARPC1B in P1 (Fig. 3a, Supplementary Fig. 6). ARPC5 expression was found to be normal in two patients with ARPC1B deficiency ( Supplementary Fig. 6). Formation of Arp2/3 complexes was quantitatively and qualitatively impaired in P1. Arp2/3 complexes containing ARPC5 were not present in P1 but were detected in HC fibroblasts lysates. Arp2/3 complexes containing ARPC2 were present in P1 at lower expression levels and with higher electrophoretic mobility relative to HC (Fig. 3b). Immunofluorescence microscopy evaluation of P1's early adherent fibroblasts revealed a high number of filopodia (elongated, fingerlike actin protrusions), whereas HC cells predominantly formed lamellipodia (flat, sheet-like actin projections) (Fig. 4a). Continuous impedance monitoring of P1's fibroblasts showed lower spreading speed compared to HC's cells (Fig. 4a). Treatment of HC's fibroblasts with the Arp2/3 inhibitor CK-666 resulted in reduced spreading capacity mimicking P1's fibroblasts behavior ( Supplementary Fig. 7). A similar pattern of aberrant spreading characterized by enrichment of filopodia and absence of lamellipodia was observed in P1's neutrophils (Fig. 4b, Supplementary Fig. 8, and Supplementary Movie 1). Real-time sequential imaging revealed that P1's cells covered a smaller area over time compared to HC's neutrophils (Fig. 4b). Cell motility was assessed in P1's fibroblasts via a time course wound healing assay. P1's fibroblasts were slower than HC's cells to close the in-vitro wound gap (Fig. 4c). Migration of neutrophils was assessed in response to Nformylmethionyl-leucyl-phenylalanine (fMLF). Directed migration was severely impaired in P1's neutrophils, as no cells were able to complete migration during the observation period, unlike HC's cells ( Fig. 4d and Supplementary Movie 2).
ARPC5 staining localized to the nucleus, to scattered dots throughout the cytoplasm, and to the edge of the plasma membrane on rescued cells, compatible with expected ARPC5 subcellular localization [14][15][16] (Fig. 5c, Supplementary Fig. 9). The adhesion and spreading pattern measured by impedance monitoring improved in wild-type ARPC5 expressing cells (Fig. 5d). Restored lamellipodia formation and wound healing could also be detected in ARPC5-rescued cells (Fig. 5c, e, and Supplementary Fig. 9). Altogether, these findings confirmed disease causality for ARPC5 mutations.
Clinical phenotype, IL-6 signaling and molecular targeting P1 (and to some extent P2, despite her young age) presented with clinical features resembling both STAT3 dominant-negative (DN; Job's syndrome) and STAT3 gain-of-function (GOF) mutations. While P1's facial dysmorphism, eczema, bacterial -Staphylococcus aureus-and fungal -Candida spp., Aspergillus fumigatus-pneumonias, poor wound healing, pneumatoceles, and scoliosis were suggestive of a Job's-like syndrome; her short stature, skin manifestations, autoimmune cytopenia -Coombs+ autoimmune hemolytic anemia, thrombocytopenia-, celiac disease, autoimmune endocrinopathies -hypothyroidism-, and a Western blotting analysis of patient 1 (P1), her parents, and healthy controls (HC) T-cell blasts lysates with antibodies specific to Arp2/3 complex subunits, as indicated. Vinculin and β-tubulin were used as loading controls. b Western blotting analysis of cell extracts from P1's and HC's fibroblasts after native gel electrophoresis. ARPC5-or ARPC2-specific antibodies were used to probe the Arp2/3 complex; GAPDH was used as a loading control. Blots in this figure are representative of at least two independent experiments. The numbers below the western blotting images (a and b) represent protein expression levels, quantitatively measured in relation to healthy controls, after normalization to the loading control. Healthy controls' average value was set at 10. PAGE polyacrylamide gel electrophoresis. Source data are provided as a Source Data file.
autoimmune hepatitis were suggestive of a STAT3 GOF-like phenotype. When her cytokine responses to IL-2, IL-4, IL-7, IL-21, interferon (IFN)α and IFNγ were evaluated in T cells and monocytes, no abnormalities were detected ( Supplementary Fig. 10). Other T-cell functions such as TCR rearrangement, signaling, and microcluster formation were normal in P1's T-cell blasts . TCR signaling and migration were also normal in CRISPR/Cas9 generated ARPC5-knockout Jurkat cells ( Supplementary Fig. 15). However, signaling through IL-6, primarily dependent on STAT3 and to a lesser extent on STAT1, was markedly diminished. Phosphorylation of STAT3 in response to in-vitro IL-6 stimulation was nearly absent in P1's CD4+ T cells yet was present in response to IL-21 ( Fig. 6a). When the expression of cell surface IL-6 receptor complex (IL-6RC) components IL-6Rα and gp130 were evaluated, very low levels of IL-6Rα were detected, while gp130 expression was mildly reduced (Fig. 6b). When IL-6RC signaling was tested in P1's fibroblasts, response to IL-6 was also nearly absent, while gp130-dependent/IL-6Rα-independent responses to oncostatin-M and IL-11 were fully preserved ( Fig. 6c). Treatment of healthy controls' CD4+ T cells with CK-666 resulted in a reduction of 21% in the surface expression of IL-6Rα, and a 1.4-fold increase in soluble IL-6Rα when compared to the vehicle treated samples (Fig. 6d), suggesting that the actin defect was indeed contributing to the impaired IL-6Rα surface expression levels. The levels of transcription for both membrane-bound IL-6Rα and soluble IL-6Rα were not affected by treatment with CK-666 ( Supplementary Fig. 16). As P1 had consistently elevated CRP levels, a known IL-6-dependent readout 17 , we hypothesized that trans-signaling, an alternative model to IL-6 classical signaling reliant on the soluble form of IL-6Rα 18 , was likely enhanced. While a clinical serum cytokine panel showed modest elevation of IL-6 (29.48 pg/mL, within 85-97.5% of normal distribution), we found markedly increased levels of both soluble IL-6Rα (sIL-6Rα) and sIL-6Rα complexed with IL-6 in P1's plasma (Fig. 7a). When tested in-vitro, P1's CD4+ T cells and fibroblasts were able to phosphorylate STAT3 in response to an IL-6/IL-6Rα fusion protein, known as hyper-IL6 19 (Figs. 6c and 7b). Such response could be controlled by pretreatment of cells with anti-IL-6Rα (tocilizumab) or soluble gp130Fc 20 (Fig. 7b). P1 died before in vivo IL-6 modulation could be explored.

Discussion
Herein we describe two unrelated patients with a previously undescribed IEI associated with germline biallelic null mutations in ARPC5.
The patients presented with recurrent and severe infections, earlyonset autoimmunity, inflammation, and dysmorphisms. In addition, we show how ARPC5-dependent actin disruption can impact IL-6 signaling, that can be considered as a therapeutic molecular target for this disease.
Genetically, ARPC5 deficiency is inherited in an autosomal recessive manner with no evidence of haploinsufficiency (i.e., heterozygous parents were asymptomatic); while clinical penetrance seems complete (i.e., both individuals with biallelic mutations were symptomatic), more patients will have to be described to fully confirm disease penetrance and expressivity.
We demonstrated that ARPC5 expression was lost in patient cells, resulting in lower expression of interacting proteins ARPC1B and ARPC1A, but not of other Arp2/3 complex components. The dependence of ARPC1 isoforms on ARPC5 levels has been previously shown a Protein expression by immunoblotting of individual Arp2/3 complex subunits in lysates from P1's fibroblasts transiently transfected (efficiency 20-40%) with empty vector or a plasmid encoding wild-type (WT) ARPC5. Vinculin was used as a loading control. b Western blotting analysis after native gel electrophoresis of cell extracts from P1's and healthy control's (HC) fibroblasts expressing WT ARPC5 by lentiviral transduction. ARPC5-or ARPC2-specific antibodies were used to probe the Arp2/3 complex. GAPDH was used as a loading control. c Immunofluorescence images of representative fibroblasts from P1 transfected with WT ARPC5 (left column) versus cells transfected with empty vector (right column). Cells were seeded on a retronectin-coated surface and fixed after 120 min. d Real-time, impedance-based monitoring of P1's ARPC5-rescued fibroblasts versus ARPC5 mock-transduced cells. Impedance values are reported as cell index (CI). Curves represent the mean (±standard deviation) cell index value from four technical replicates. e Wound healing assay with WT ARPC5-rescued fibroblasts from P1 (middle column) and mock-transduced P1 cells (right column), HC cells were used as controls (left column). The gap length shown at 0 h corresponds to 0.94 mm. All results in this figure are representative of at least two independent experiments. The numbers below the western blotting images represent protein expression levels relative to WT ARPC5-rescued P1 fibroblasts (a) or HC fibroblasts (b), after normalization to the loading control. PAGE polyacrylamide gel electrophoresis. Source data are provided as a Source Data file.
in HeLa cells 21 and is now confirmed in human disease. In contrast, ARPC1B deficiency does not seem to affect ARPC5 expression levels, suggesting that ARPC5 deficiency might have a broader impact on biology and disease. Moreover, depletion of ARPC1B levels in the patient with ARPC5 deficiency supports the phenotypic overlaps between the two diseases [7][8][9][10][11] .
Functionally, the Arp2/3 complex is recognized by its unique ability to nucleate actin filaments at an angle from a preexisting filament, resulting in a branched network of polymerized actin 3 . Branched actin is the predominant form of actin organization in lamellipodia, podosomes and invadopodia, all required but not indispensable for cell motility 22 . Experimental disruption of the Arp2/3 complex results in abrogation of lamellipodial structures with enrichment of filopodia, as confirmed in P1's fibroblasts and neutrophils 23,24 . Similarly defective actin distribution patterns have been documented in platelets, neutrophils, and T cells from patients with ARPC1B deficiency 7,8,10 , as well as in patients with HEM-1 deficiency 6 , a protein required to activate the Arp2/3 complex to generate lamellipodia 25 .
We showed that fibroblast migration was impaired in ARPC5 deficiency. This finding correlated with the poor and delayed wound healing experienced by our patients. Impaired wound healing was also observed with in-vitro testing of murine Arpc3 −/− fibroblasts 24 and Arpc5-silenced rat smooth muscle cells 26 . Interestingly, migration of fibroblasts from a patient with ARPC1B deficiency was unaffected 8 , suggesting that not all Arp2/3-related defects behave as phenocopies in the same cell lineage and function. Moreover, while fibroblast defects are certainly contributing to the wound healing defect, they are unlikely to be the only cause: the hyperinflammatory state and impaired neutrophil migration, both seen in other PID/IEI with impaired wound healing, must be considered as other likely responsible factors 27,28 . Thrombocytopenia in ARPC5 deficiency is probably another multifactorial complication in this disease. Despite peripheral blood thrombocytopenia, bone marrow biopsies on P1 never showed increased megakaryocytes but did present with dysplastic features, similarly to what was reported in patients with ARPC1B deficiency 7 , which is also markedly diminished in ARPC5 deficiency. Her persistent hepatosplenomegaly and broad autoimmunity profile may have also potentially contributed to platelet sequestration and thrombocytopenia.
Although quantitatively decreased and qualitatively altered, the assembly of Arp2/3 complexes was not completely abrogated Data are representative from two independent experiments. d Enriched CD4 T cells were treated either with a vehicle (DMSO) or Arp2/3 complex inhibitor CK-666 (100 mM) for 22 h. Flow analysis was used to evaluate the surface expression of IL-6Rα. The supernatants of the same samples treated with CK-666 were collected, and the Luminex assay was utilized to determine the concentration of soluble IL-6Rα. The IL-6Rα expression levels were calculated relative to the vehicle treated controls. Data are expressed as mean + standard deviation (SD) from five different healthy controls. Source data are provided as a Source Data file.
in the context of ARPC5 deficiency. The same was seen in platelet lysates from patients with ARPC1B deficiency 7 and likely contribute to the non-embryonic lethality of these genotypes 22 . Whether Arp2/3 complex integrity in these scenarios is maintained by alternative isoforms of ARPC1 (as reported by Leung et al.) 29 and ARPC5, or by non-canonical conformations of the complex lacking these subunits 30 , is yet to be established. Of note, different compositions of the Arp2/3 complex have been shown to nucleate actin with distinct efficiency and to present variable stability. Specific combinations of the complex are likely suited to particular physiologic processes 21,30 . In this setting, Faessler et al., very recently reported how murine and rat ArpC5 subunit isoforms (i.e., Arpc5 and Arpc5L) can alternatively and differently regulate particular Arp2/3-dependant functions. Interestingly, some differences became evident between human vs. rodent species as ARPC5 deficiency in our work does not seem to affect ARPC5L accumulation, while ArpC5 deficiency consistently increased the ArpC5L levels likely affecting the functions dependent on each type of Arp2/3 complexes 16 . Not surprisingly, the data presented here showed that depriving cells of the ARPC5 subunit resulted in dysregulation of several but not all Arp2/3-dependent functions. Altogether, these features help to explain why signaling through IL-6Rα but not all surfaceexpressed cytokine receptors are affected by ARPC5 deficiency. Interestingly, in depth analysis of the impact of ARPC5 deficiency on other T-cell functions failed to detect deleterious consequences as previously demonstrated in ARPC1B deficiency 10,31 .
Despite the inherent limitations of studying rare diseases with small number of patients 32 , we showed that two unrelated patients carrying biallelic null mutations in ARPC5 suffered from a previously unreported disease. While the association between ARPC5 deficiency and CNS or other syndromic manifestations will be further clarified as more patients are described, we efficiently proved that the ARPC5 protein was not expressed, Arp2/3 complex formation was impaired, and subsequently different actin-related functions were variably affected in specific cell lineages in this disease. Rescue of ARPC5 expression reestablished Arp2/3 complex conformation and function. As part of the clinical-pathophysiological evaluation, we also showed that the IL-6 pathway was distinctively impacted in this disease. IL-6 signaling was tested, trans-signaling seemed to have a critical role in the disease and offered a molecular target for treatment. This work emphasizes the importance of studying rare genetic diseases through rigorous methodological criteria to unveil the underlying pathophysiology mechanism and potential therapeutic opportunities.

Patients and samples
The patients' legal representatives provided informed consent at their respective institutions, in accordance with the Declaration of Helsinki, under protocols approved by local institutional review boards at the Rockefeller University, and/or National Institutes of Health (NIH)/ National Institute of Allergy and Infectious Diseases (NIAID) protocol 10-I-0216; ClinicalTrials.gov Identifier: NCT01222741. The patients' legal representatives consented to the publication of medical information and images related to the cases. Samples from Patient 1's family were collected at several time points between 2019 and 2021 at Dalhousie University, Halifax, Nova Scotia, Canada and sent to NIH for genetic and functional studies; Patient 2's family samples were collected at different timepoints between 2012 and 2013 at the Children's Medical Center, Tehran, Iran and sent to the Rockefeller University for genetic testing. Blood samples and biopsies from patients, family members, and healthy donors were obtained following standard of care under approved protocols by the National Institutes of Health institutional review board. Results are shown as mean + SEM. Data generated from two independent experiments, performed in duplicate each time. b IL-6 trans-signaling modulation in P1's CD4+ T cells. P1's cells were pretreated with soluble gp130Fc (1 µg/mL) or tocilizumab (10 µg/mL) for 90 min. A subset of cells was left untreated to serve as control. Then, cells were stimulated with hyper IL-6 (100 ng/mL) for 20 min. A subset of cells was not stimulated to serve as baseline. Sequentially, phosphorylation of STAT3 was measured by flow cytometry (upper panel). The bar graph shows soluble gp130Fc and tocilizumab pretreatment effect on STAT3 phosphorylation in response to hyper IL-6 stimulation compared to untreated (−), stimulated cells (lower panel). This experiment was conducted once. Source data are provided as a Source Data file.

Sanger sequencing
Selected next-generation sequencing results were confirmed and carrier testing was performed by Sanger sequencing. Genomic DNA was PCR-amplified using M13-tagged specific variant-flanking primers, and the PCR products were subjected to Sanger sequencing using M13primers and the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Cat. 4337452) according to the manufacturer's protocol. The Sanger sequencing data was analyzed using DNAStar Lasergene 16 SeqMan.
Fluorescence microscopy of fibroblasts P1's or HC's fibroblasts were seeded in 6-well plates pre-coated with RetroNectin (Takara, Cat. T100B) in complete DMEM medium and allowed to spread in a humidified incubator at 37°C with 5% CO 2 . After specified times, cells were washed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 15 min, washed again, then permeabilized in 0.1% Triton X-100 in PBS for 10 min and blocked (10% FBS and 0.1% Triton X-100 in PBS) for 30 min. Cells were then incubated in blocking buffer with either Alexa Fluor 488 Phalloidin (Invitrogen, Cat. A12379), mouse anti-HA (Cell Signaling technology, Cat. 2367), or rabbit anti-Flag (Cell Signaling Technology, Cat. 14793 S) antibodies. Next, cells were washed in PBS. Samples stained with anti-HA and anti-Flag antibodies were incubated for 1 h with Alexa Fluor 594 or Alexa Fluor 568-conjugated secondary antibodies in blocking buffer (Invitrogen, Cat. A11072 and A21069). After washes, all samples were incubated with DAPI (Cell Signaling Technology, Cat. 4083) in PBS, for 10 min. Samples were washed twice in PBS and ready for imaging. Filopodia formation was best visualized 20 min after cell seeding and lamellipodia formation 120 min after cell seeding; therefore, those time points were selected for imaging. Images were collected using a ZOE fluorescent cell imager (Bio-Rad).

Real-time cell analysis of adhesion and spreading
The rate and magnitude of adherence of fibroblasts was measured with the xCELLigence RTCA MP (Agilent, Santa Clara, CA). Briefly, E-plates 96 (Agilent, Santa Clara, CA) were background normalized after equilibrating the wells with 100 µL of complete media for 30 min. Following this step, 1 × 10 4 HC's or P1's fibroblasts were seeded and immediately placed onto the xCELLigence RTCA MP machine at 37°C with 5% CO 2 to begin recording impedance every 1-2 min for 5 h and every 15 min thereafter. Impedance values were converted into the cell index by the RTCA software (Agilent, Santa Clara, CA) and plotted over time using the GraphPad Prism software version 8.3.0 (GraphPad, LLC). To measure spreading of neutrophils, isolated polymorphonuclear neutrophils (1 × 10 6 cells/ml in 20 µL HBSS with Ca 2+ and Mg 2+ buffer (Gibco, Cat. 14025134) were added on the center of microscope slide. A coverslip was placed over the cells and digital images were captured at five second intervals for 600 s. The area and perimeter of the cells were measured using Infinity Analyze software (Lumenera, version 5.0.3).

Cell migration assays
Fibroblasts migration was assessed with a wound healing (scratch) assay. Fibroblasts were seeded in a 12-well plate and serum-starved overnight. The next day, a scratch in the cell monolayer was generated with a pipette tip (20 μl tip, tip width 0.94 mm, Thermo Scientific Art tips). Cells were washed 2x in PBS and from then on cultured in DMEM supplemented with 2% FBS and 2 mM L-glutamine. Cells were imaged right after scratching (t = 0), at 8 h, and 24 h after the scratch was made. Images were captured with a Lionheart FX Automated Live Cell Imager (Agilent BioTeK) using a 4× objective with the high contrast brightfield accessory. To improve visualization, digital phase contrast processing was applied to entire images using Gen5 Image+ software (Agilent BioTeK). Neutrophil chemotaxis was monitored across a 260 µm platform separating the "cell" well from the "chemoattractant" well using the EZ-TAXIScan (Effector Cell Institute, Tokyo, Japan). Isolated neutrophils (5 × 10 3 cells in 1.0 µl) were added to the "cell" well of the EZ-TAXIScan and 1.0 µl of either buffer (0.1% BSA in RPMI, 20 mM HEPES) or 5 × 10 −8 M fMLF (N-formylmethionyl-leucyl-phenylalanine, Sigma-Aldrich, Cat. F3506) was added to the opposing "chemoattractant" well. Digital images of the migrating PMNs were captured every 30 s for 1 h. Images were converted to stacks using the ImageJ software (version 1.53t; NIH). Ten randomly selected cells were electronically traced using the ImageJ plug-in, MTrackJ and the sequential positional coordinates of individual migrating cells were determined as a function of time. The tracks of individual migrating cells were reconstructed and plotted with the position of each cell anchored at the origin at t = 0. Since data were collected with time and position, multiple parameters could be derivedoverall distance, directed distance (parallel to the chemoattractant, random distance (orthogonal to the chemoattractant), overall velocity, directed and random velocity vectors, and time-to-event analysis (number of cells completing migration and elapsed time). To assess migration in Jurkat cells, 5 × 10 5 wild-type (WT) or ARPC5-KO cells were suspended in 100 μL of RPMI 1640 media supplemented with 10% FBS (RPMI complete). The cells were loaded into 5.0 μm Transwell inserts (Corning) which were placed onto 24-well plates containing 450 μL of RPMI complete with or without 800 ng/mL CXCL12 (Peprotech). After 3 h of incubation at 37°C, cells that had migrated to the lower chamber were collected and counted using an automatic cell counter (Countess II FL, ThermoFisher Scientific). Results were calculated as percentage of cells that had migrated out of total input cells.

Flow cytometry
For identification of surface proteins, single-cell suspensions of washed PBMC were stained with fluorochrome-conjugated antibodies (see list below) for 30 min at 4°C. Then, cells were washed with stain buffer (BD Biosciences, Cat. 554656) twice and were ready for acquisition. For detection of the intracellular proteins FOXP3 and T-bet, cells were fixed

Data availability
Whole exome sequencing datasets generated and analyzed during the current study are available in NCBI's Sequence Read Archive with accession no. PRJNA889418. Single-cell RNA sequencing raw and processed data have been deposited in NCBI's GEO data repository with accession no. GSE215451. T-cell receptor β sequencing data have been deposited in the ImmuneACCESS database [https://doi.org/10.21417/ CJNS2023NC]. Source data are provided with this paper.